Antibiotic substance from Actinoplanes sarveparensis CBS 305.76 (antibiotic L 13365)

ABSTRACT

A new antibiotic prepared from aerobic cultivation of Actinoplanes sarveparensis and a method for producing the same from a culture medium.

SUMMARY OF THE INVENTION

The present invention refers to a new antibiotic substance obtained bythe fermentation of a strain belonging to the genus Actinoplanes,specifically Actinoplanes sarveparensis. The novel antibiotic substance,hereinafter referred to as antibiotic L 13365 is a light yellowcrystalline compound having characteristic identifiable properties suchas melting point, infrared and ultraviolet absorption maxima.

As stated above, antibiotic L 13365 is produced by cultivation of afermenting strain named Actinoplanes sarveparensis. This strain,identified with out collection number A/13826, was isolated from a soilsample collected at Sarvepar Village, India. The strain has beendeposited and made part of the stock culture collection of CBS (CentraalBureau Voor Schimmelcultures -- Oosterstraat 1 -- Baarn -- TheNetherlands) where it was assigned the number 305.76.

In the preparation of antibiotic L 13365, the microorganism iscultivated under aerobic conditions in an aqueous nutrient mediumcontaining an assimilable source of carbon, an assimilable source ofnitrogen and inorganic salts.

As used herein, the term "assimilable source" refers to a source of asubstance required for the growth of the organism or for the productionof the antibiotic supplied in a form which may be absorbed and used bythe organism.

Ordinarily, the antibiotic producing strain is precultured in a shakeflask until substantial antibiotic activity is present then the cultureis used to inoculate jar fermentors containing a nutrient fermentativemedium.

The culture is normally incubated at from about 25°-35° C under aerobicconditions for a time sufficient to produce a substantial antibioticlevel. During this time, microbiological assays are carried out by theagar diffusion method to control the concentration of the antibioticsubstance produced.

After eliminating the mycelium cake by filtration, the antibioticsubstance is recovered from the filtered fermentation broth byconventional procedures known to the art, such as, for instance, byextraction with an organic solvent in which the antibiotic substance issoluble and which is immiscible with the aqueous medium. The extractionis carried out after adjustment of the pH of the filtrate between about2 to about 4. Suitable organic solvents for the extraction areadvantageously selected from alkanols containing from 1 to about 6carbon atoms, (C₁ -C₄)alkyl esters of lower aliphatic acids, or lowerhalogenated hydrocarbons. The solvent may then be separated from thefermentation broth by high-speed centrifugation, concentrated to about1/200-1/400 of its original volume, cooled and allowed to stand until aprecipitate forms which may be recovered by filtration. This precipitateconsists of antibiotic L 13365 substantially free of major impurities.The mother liquors are collected and poured into an excess of an inertnon-polar solvent such as light petroleum or the like giving a furtheramount of crude antibiotic L 13365. The crude product thus obtained isdissolved in a methanol-acetone mixture and is precipitated from thesolution by addition of acidic water.

The two portions are gathered together and may be further purified bycolumn chromatography using a chloroform-methanol mixture as the elutingsystem.

Antibiotic L 13365 is finally crystallized from methanol:ethyl acetatehaving a 1:1 ratio.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a typical ultraviolet spectrum of antibiotic L 13365 asrecorded from a solution of methyl cellosolve.

FIG. 2 represents a typical infrared spectrum of antibiotic L 13365.

DETAILED DESCRIPTION OF THE INVENTION

The antibiotic substance L 13365 shows a remarkable antibacterial invitro and in vivo activity. More particularly, antibiotic L 13365exhibits an outstanding in vitro antimicrobial action, especiallyagainst gram-positive bacteria, as shown in Table I below.

                  TABLE I                                                         ______________________________________                                                             Minimal Inhibitory                                       Strain               Concentration (μg/ml)                                 ______________________________________                                        Staphylococcus aureus ATCC 6538                                                                    0.025                                                    Staphylococcus aureus Tour                                                                         0.025                                                    Streptococcus haemolyticus C 203                                                                   0.0062                                                   Diplococcus pneumoniae UC 41                                                                       0.00078                                                  Clostridium perfingens ISS 30543                                                                   0.012                                                    Mycoplasma gallisepticum H21 CZB                                                                   0.2                                                      ______________________________________                                    

Furthermore, as stated above, antibiotic L 13365 also displays anexcellent in vivo activity against experimental infections in mice. Moreparticularly, the dose effective to protect 50 percent of the mice(ED₅₀) using antibiotic L 13365 against experimental infections provokedby Staphylococcus aureus, Streptococcus haemolyticus and Diplococcuspneumoniae are listed hereinbelow in Table II.

                  TABLE II                                                        ______________________________________                                        Infection Strain      ED.sub.50 mg/kg* s.c.                                   ______________________________________                                        Streptococcus haemolyticus                                                                          0.2                                                     Staphylococcus aureus 10                                                      Diplococcus pnemoniae 0.4                                                     ______________________________________                                         *median effective dose (ED.sub.50)                                       

The new antibiotic substance is active also against microorganismstrains which are resistant to other commonly used antibiotics.Representative examples of the minimum inhibiting concentrations (MIC)of antibiotic L 13365 against Staphylococcus aureus strains resistant toseveral antibiotics are reported in Table III.

                                      TABLE III                                   __________________________________________________________________________    Strain         MIC of Other Antibiotics                                                                    MIC of Antibiotic L 13365                        __________________________________________________________________________    Staphylococcus aureus                                                                         penicillin >100                                                                            0.4                                              ATCC 6538 resistant to                                                        penicillin                                                                    Staphylococcus aureus Tour                                                                    streptomycin >100                                                                          0.078                                            resistant to streptomycin                                                     Staphylococcus aureus                                                                         tetracycline >100                                                                          0.15                                             ATCC 6538 resistant to                                                        tetracycline                                                                  Staphylococcus aureus                                                                         rifampicin >100                                                                            0.01                                             ATCC 6538 resistant to                                                        refampicin                                                                    Staphylococcus aureus                                                                         neomycin >100                                                                              0.005                                            ATCC 6538 resistant to                                                        neomycin                                                                      Staphylococcus aureus                                                                         erythromycin >100                                                                          0.04                                             ATCC 6538 resistant to                                                        erythromycin                                                                  Staphylococcus aureus                                                                         chloramphenicol >100                                                                       0.02                                             ATCC 6538 resistant to                                                        chloramphenicol                                                               Staphylococcus aureus                                                                         cephaloridine >100                                                                         0.078                                            ATCC 6538 resistant to                                                        cephaloridine                                                                 Staphylococcus aureus                                                                         kanamycin >100                                                                             0.04                                             ATCC 6538 resistant to                                                        kanamycin                                                                     Staphylococcus aureus                                                                         bacitracin >100                                                                            0.02                                             ATCC 6538 resistant to                                                        bacitracin                                                                    Staphylococcus aureus                                                                         lincomycin >100                                                                            0.15                                             ATCC 6538 resistant to                                                        lincomycin                                                                    __________________________________________________________________________

The toxicity of antibiotic L 13365, administered either orally orintraperitoneally to mice is very low with the median lethal dose (LD₅₀)values higher than 1000 mg/kg.

Description of Actinoplanes sarveparensis A/13826 Macroscopicexamination of colonies

The strain grows well on various nutrient agars. In oatmeal agar, thecolonies are 3 to 4 mm in diameter, show irregular contours and a roughsurface. Aerial mycelium is always absent.

Microscopic examination

Sporangia are produced in several agar media. They have a regularglobose shape with a diameter ranging from 13 to 20 μ. The zoospores,highly motile, are spherical but occasionally are slightly elongatedwith a diameter of 1.5-2 μ. On the basis of these characteristics, thestrain A/13826 is ascribed to the genus Actinoplanes and given the nameActinoplanes sarveparensis CBS 305.76. Table IV reports the culturalcharacteristics of Actinoplanes sarveparensis CBS 305.76 cultivated onvarious standard media suggested by Shirling and Gottlieb (Intern. J.Syst. Bact., 16, 313-340, 1966) and other media recommended by Waksman(The Actinomycetes, Vol. II, The Williams and Wilkins Co., 1961). Thecultural characteristics were determined after 6-14 days of incubationat 30° C.

Table V reports the utilization of carbon sources examined according tothe method of Pridham and Gottlieb (Intern. J. Syst. Bact. 56, 107,1948).

Table VI reports the physiological characteristics of the strain.

                  TABLE IV                                                        ______________________________________                                        Cultural Characteristics of Actinoplanes sarveparensis                        CBS 305.76                                                                    Culture Medium     Cultural Characteristics                                   ______________________________________                                        Medium No. 2     Abundant growth, wrinkled                                    (yeast extract-malt agar)                                                                      surface, orange to brown; (cen-                                               tral part of colony is darker).                              Medium No. 3     Scant growth, thin, hyaline to                               (oatmeal agar)   light orange - production of                                                  sporangia.                                                   Medium No. 4     Moderate growth, crusty surface,                             (inorganic salts-starch agar)                                                                  light orange (9/G/7).                                        Medium No. 5     Abundant growth, crusty surface,                             (glycerol-asparagine agar)                                                                     brilliant orange (9/I/11).                                   Medium No. 6     Scant growth, crusty suface,                                 (peptone-yeast extract-                                                                        brown (13/C/7).                                              iron agar)                                                                    Medium No. 7     Moderate growth, crusty surface,                             (tyrosine agar)  orange (11/F/8).                                             Oatmeal agar     Abundant growth, rough surface,                              (according to Waksman)                                                                         orange (9/H/12), (central part                                                of colony is darker).                                        Hyckey and Tresner's agar                                                                      Moderate growth, wrinkled                                                     surface brown (18/A/12).                                     Czapek glucose agar                                                                            Abundant growth, wrinkled                                                     surface orange (11/G/11).                                    Glucose asparagine agar                                                                        Abundant growth, rough surface,                                               brilliant orange (9/I/11).                                   Nutrient agar    Moderate growth, rough surface,                                               orange (11/F/8).                                             Potato agar      Moderate growth, wrinkled                                                     surface, orange to brown - pro-                                               duction of sporangia.                                        Bennett's agar   Moderate growth, wrinkled                                                     surface, light orange (9/D/5).                               Calcium malate agar                                                                            Scant growth, thin hyaline to                                                 light orange - production of                                                  sporangia.                                                   Skim milk agar   Abundant growth, wrinkled                                                     surface, orange to light brown,                                               patch shadows.                                               Czapek agar      Abundant growth, wrinkled                                                     surface, orange (11/G/11).                                   Egg agar         Scant growth, rough surface,                                                  hyaline.                                                     Pept. glucose agar                                                                             Moderate growth, wrinkled                                                     surface, deep orange (11/B/12).                              Agar             Very scant growth, thin, hyaline                             Loeffler Serum   Very scant growth, orange                                    Potato           Scant growth, wrinkled, deep                                                  orange                                                       Gelatin          Scant growth, light orange                                   Cellulose        No growth                                                    ______________________________________                                    

Color determination was made by the method of Maerz and Paul (Maerz, A.and M. Reg. Paul 1950. A dictionary of color, 2nd ed. M. Grow -- HillBook Company, Inc., New York).

The numbers of some culture media refer to those given according toShirling and Gottlieb (Intern. J. Syst. Bact., 16, 313-340, 1966).

                  TABLE V                                                         ______________________________________                                        Utilization of Carbon Compounds                                               Carbon Sources       Utilization                                              C.sub.5 Arabinose    +                                                        Xylose               +                                                        C.sub.6 Glucose      +                                                        Fructose             +                                                        Mannose              +                                                        Mannitol             +                                                        Inositol             +                                                        Rhamnose             +                                                        (C.sub.6).sub.2 Sucrose                                                                            +                                                        Lactose              +                                                        (C.sub.6).sub.3 Raffinose                                                                          -                                                        (C.sub.6).sub.4 Cellulose                                                                          -                                                        Special Salicin      -                                                        ______________________________________                                         + means utilization                                                           - means lack of utilization                                              

                  TABLE VI                                                        ______________________________________                                        Physiological Characteristics                                                         Test            Results                                               ______________________________________                                        Hydrolysis of starch    positive                                              H.sub.2 S formation     positive                                              Melanin production      negative                                              Tyrosinase reaction     negative                                              Casein hydrolysis       positive                                              Calcium malate hydrolysis                                                                             positive                                              Nitrate reduction       positive                                              Litmus milk coagulation negative                                              Litmus milk peptonization                                                                             positive                                              Gelatin liquefaction    positive                                              ______________________________________                                    

Production and Isolation of the Antibiotic Substance

A preferred method for producing the antibiotic substance is byaerobically pre-culturing the Actinoplanes sarveparensis CBS 305.76 in anutrient medium until substantial antibiotic activity is present at a pHvalue ranging from about 6 to about 10. The following shake flaskculture was found satisfactory in the practice of the present invention.

    ______________________________________                                        Meat extract         3.0 g/l                                                  Tryptone             5.0 g/l                                                  Yeast extract        5.0 g/l                                                  Glucose              1.0 g/l                                                  Soluble starch       24.0 g/l                                                 Calcium carbonate    4.0 g/l                                                  Distilled water      q.s. to 1000 ml                                          ______________________________________                                    

The flakes are shaken for about 24 hours at about 28°-30° C and thepre-cultures (one liter) are used to inoculate jar fermentors eachcontaining 10 liters of the following nutrient medium:

    ______________________________________                                        Meat extract         40 g                                                     Peptone              40 g                                                     Yeast extract        10 g                                                     Sodium chloride      25 g                                                     Soybean meal         100 g                                                    Glucose              500 g                                                    Calcium carbonate    50 g                                                     Tap water            q.s. to 10 liters                                        ______________________________________                                    

The fermentation batches are incubated aerobically under stirring at28°-30° C. At intervals, the antibiotic activity is assayedmicrobiologically by the agar diffusion method using Staphylococcusaureus as the test organism. The maximum activity will be reached afterabout 72-96 hours of fermentation.

Isolation and Purification of Antibiotic L 13365

One method of isolating the enzyme found to be satisfactory was asfollows: the fermentation broth (80 liters) was filtered using 1 percentclarcel (W/V) as a filter aid. The mycelium cake is discarded and thefiltered solution, acidified to pH 2.5 with 10 percent HCl, is extractedtwice with an amount of ethyl acetate corresponding to about 50 percentof its volume.

The organic phase was separated from the aqueous one by means ofhigh-speed centrifugation, then dried over Na₂ SO₄, concentrated at45°-50° C under vacuum to about 1/300 of its orginal volume and finallycooled to about 0°-10° C.

A crude precipitate formed, which was collected on a filter, washed witha small quantity of ethyl acetate and dried at about 45° C under vacuum.1.140 Grams of the antibiotic substance L 13365 were obtained with apurity degree of about 70 percent, determined spectrophotometrically.

The mother liquors deriving from the above filtration were poured into20 volumes of light petroleum and an additional amount (2.550 g) ofantibiotic substance with a purity degree of 20-25 percent (determinedspectrotometrically) was obtained. This substance was suspended in asmall amount of methanol:acetone 9:1 mixture, filtered from anyinsoluble impurities and diluted with water: the solution was brought toa pH value of about 2.5 by means of 10 percent HCl under stirring andthe precipitate which formed was collected by centrifugation andredissolved in a minimum amount of butanol at about 45°-50° C. Thesolution was concentrated under vacuum to about 1/5 of the originalvolume and cooled to about 4° C. The resulting precipitate, after beingcollected and dried under vacuum, was the antibiotic substance L 13365(0.450 g) having a purity degree of about 80 percent (determinedspectrophotometrically). The obtained crops were then subjected tocommon purification operations known to the art. To this purpose, theywere dissolved in a minimum amount of a chloroform:methanol 85:15mixture and chromatographed through a silica-celite column (1:1 V/V),previously activated at 100° C, and washed with the abovechloroform/methanol mixture.

Elution and thin layer chromatography control of the fractions areperformed with the same mixture. The fractions collected according tot.l.c. analysis data were concentrated under vacuum to a small volume.Upon adding diethyl ether, a precipitate formed, consisting ofantibiotic L 13365 with a purity degree of about 95 percent (determinedspectrophotometrically). Said precipitate was dissolved in amethanol:ethyl acetate 1:1 mixture, heated to 45° C and filtered fromany insolubles; upon cooling to 4° C and standing overnight at the sametemperature substantially pure antibiotic L 13365 was obtained a lightyellow needles.

Chemico-physical Properties of Antibiotic L 13365

Antibiotic L 13365 is a light yellow crystalline powder with acidiccharacter. Analysis of an acid hydrolisate of antibiotic L 13365 in 6Nhydrochloric acid in a sealed tube at 110° C revealed four amino acids,three of them have been identified in an amino acid autoanalyzer asaspartic acid, threonine and glycine. Furthermore, antibiotic L 13365 ischaracterized by the following properties:

(1) Melting point: -- 210° C

(2) Elemental analysis: -- C = 51.35, H = 5.05, N = 10.50, S = 11.05 andO (by difference) = 22.05.

(3) Ultraviolet and visible absorption spectrum

    ______________________________________                                        Solvent        λ.sub.max (nm)                                                                      E.sup.1%.sub.1cm                                  ______________________________________                                        Hydrochloric acid 0.1N                                                                       360          103                                                              290 (shoulder)                                                                237          326                                               Buffer 0.15 M  408 (shoulder)                                                 pH 7.5         378          67                                                               290 (shoulder)                                                                237          326                                               Buffer 0.15 M  408          99                                                pH 8.8         290 (shoulder)                                                                238          336                                               ______________________________________                                    

Ultraviolet absorption spectra were determined with a UNICAM Sp 800ultraviolet spectrophotometer using solutions of L 13365 inmethylcellosolve (MCS)-buffers at different pH in one to one proportion.The complete ultraviolet spectrum is shown in FIG. 1.

(4) Fluorescence spectrum: -- The antibiotic contains a stronglyfluorescent chromophore and a solution of the product in 0.1 N sodiumhydroxide excited at 240 nm shows an emission spectrum with maxima at355 and 490 nm. Fluorescense spectrum was determined with a Perkin ElmerMod. MPF - 44 fluorescence spectrophotometer.

(59 Infrared spectrum: -- Characteristic absorption bands in nujol havebeen observed at the following frequencies (cm⁻¹): 3400 (shoulder), 3350(sharp), 3200 (shoulder), 3100 (sharp), 2930 and 2870 (nujol),2800-2400, 2380 (atmospheric carbon dioxide), 1750 (sharp), 1670(sharp), 1620 (sharp), 1540 (sharp), 1480 (sharp), 1460 and 1380(nujol), 1420 (sharp), 1340 (sharp), 1320 (sharp), 1280 (sharp), 1240(sharp), 1195 (sharp), 1170 (sharp), 1145 (sharp), 1120 (sharp), 1075(broad), 1015 (sharp), 990 (sharp), 935 (sharp), 920 (broad), 900(sharp), 865 (sharp), 840 (sharp), 800 (sharp), 770 (sharp), 755(sharp), 740 (sharp), 720 (nujol). The infrared spectrum was determinedwith a Perkin Elmer Mod. 157 spectrophotometer. The complete infraredspectrum is shown in FIG. 2.

(6) Specific rotation: -- [α]₄₃₆.sup. 25 = + 125° (C = 0.8 percent inmethanol:chloroform 1:1)

(7) Solubility: -- The compound is soluble in dimethylsulfoxide,dimethylformamide and in chloroform/-methanol mixtures; slightly solublein chloroform, methanol, methanol/ethyl acetate mixtures, sodiumbicarbonate solutions and glacial acetic acid; insoluble in water and inthe other common organic solvents.

(8) Characteristic reactions

    ______________________________________                                        Fehling             positive                                                  Tollens             positive                                                  KMnO.sub.4          positive                                                  H.sub.2 SO.sub.4 conc.                                                                            dark brown color                                          Ninhydrin           negative                                                  FeCl.sub.3          positive                                                  Millon              negative                                                  Schiff              negative                                                  Anthrone            positive                                                  Folin Ciocalteau    negative                                                  Elson Morgan        negative                                                  ______________________________________                                    

(9) Acidity: An ionizable function with pK_(a) 7.8 is evidenced bypotentiometric titration with 0.1 N sodium hydroxide of antibiotic L13365 in a 2-methoxyethanol:water 4:1 solution. The equivalent weightdetermined accordingly is 1400.

(10) Chromatographic behavior

    ______________________________________                                        Solvent System            Rf*                                                 ______________________________________                                        Buffer pH 6.0 saturated n-butanol                                                                       0.75                                                Water saturated n-butanol + 2%                                                                          0.80                                                p-toluenesulfonic acid                                                        Water saturated n-butanol + 2%                                                                          0.70                                                conc. ammonia                                                                 Butanol saturated buffer pH 6.0                                                                         0.0                                                 Ammonium chloride (20% W/V in water)                                                                    0.0                                                 n-butaniol:methanol:water 40:10:20                                                                      0.90                                                containing 0.75 g methyl orange                                               n-butaol:methanol:water 40:10:30                                                                        0.90                                                water:acetone 1:1         0.35                                                water saturated ethyl acetate                                                                           0.0-0.31                                            chloroform:methanol 85:15 (**)                                                                          0.51                                                ______________________________________                                         (*) = Paper chromatography on Whatman paper no. 1. Antibiotic visualized      on agar plates seeded with S. aureus.                                         (**) = Tlc on silica gel plates HF/UV.sub.254. Fluorescent spot under         ultra-violet light.                                                      

What is claimed is:
 1. Antibiotic L 13365, said antibiotic having thefollowing characteristics:(a) Melting point: 210° C; (b) Approximateelemental composition of 51.35 percent carbon, 5.05 percent hydrogen,10.50 percent nitrogen, 11.05 percent sulfur and 22.05 percent oxygen;(c) Ultraviolet spectrum: characteristic ultraviolet absorption bands inthe following solvent systems

    ______________________________________                                        Solvent        λ.sub.max (nm)                                                                      E.sup.1%.sub.1cm                                  ______________________________________                                        Hydrochloric acid 0.1N                                                                       360          103                                                              290 (shoulder)                                                                237          326                                               Buffer 0.15 M  408 (shoulder)                                                 pH 7.5         378          67                                                               290 (shoulder)                                                                237          326                                               Buffer 0.15 M  408          99                                                pH 8.8         290 (shoulder)                                                                238          336                                               ______________________________________                                    

(d) Infrared spectrum: characteristic infrared absorption bands in nujolat the following frequencies (cm⁻¹): 3400 (shoulder), 3350, 3200(shoulder), 3100 (sharp), 2930 and 2870 (nujol), 2800-2400, 2380(atmospheric carbon dioxide), 1750 (sharp), 1670 (sharp), 1620 (sharp),1540 (sharp), 1480 (sharp), 1460 and 1380 (nujol), 1420 (sharp), 1340(sharp), 1320 (sharp), 1280 (sharp), 1240 (sharp), 1195 (sharp), 1170(sharp), 1145 (sharp), 1120 (sharp), 1075 (broad), 1015 (sharp), 990(sharp), 935 (sharp), 920 (broad), 900 (sharp), 865 (sharp), 840(sharp), 800 (sharp), 770 (sharp), 755 (sharp), 740 (sharp), 720(nujol); (e) Specific rotation: -- α]₄₃₆ ²⁵ = +125° (C = 0.8 percent inmethanol:chloroform 1:1); (f) Solubility: -- Soluble indimethylsulfoxide, dimethylformamide and in chloroform/methanolmixtures. Slightly soluble in chloroform, methanol, methanol/ethylacetate mixtures, sodium bicarbonate solutions and glacial acetic acid;insoluble in water and in the other common organic solvents; (g)Characteristic reactions:

    ______________________________________                                        Fehling             positive                                                  Tollens             positive                                                  KMnO.sub.4          positive                                                  H.sub.2 SO.sub.4 conc.                                                                            dark brown color                                          Ninhydrin           negative                                                  FeCl.sub.3          positive                                                  Millon              negative                                                  Schiff              negative                                                  Anthrone            positive                                                  Folin Ciocalteau    negative                                                  Elson Morgan        negative                                                  ______________________________________                                    

(h) Ionizable functions: an ionizable function potentiometricallyevidenced with pK_(a) = 7.8 (2-methoxyethanol:water 4:1); (i) R.F.values: chromatography on Whatman paper No. 1; Visualization of thespots by microbiological development on Staphylococcus aureus

    ______________________________________                                        Eluting System            Rf value                                            ______________________________________                                        Buffer pH 6.0 saturated n-butanol                                                                       0.75                                                Water saturated n-butanol + 2%                                                                          0.80                                                p-toluenesulfonic acid                                                        Water saturated n-butanol + 2%                                                                          0.70                                                conc. ammonia                                                                 Butanol saturated buffer pH 6.0                                                                         0.0                                                 Ammonium chloride (20% W/V in water)                                                                    0.0                                                 n-butanol:methanol:water 40:10:20                                                                       0.90                                                containing 0.75 g methyl orange                                               n-butanol-methanol:water 40:10:30                                                                       0.90                                                water:acetone 1:1         0.35                                                water saturated ethyl acetate                                                                           0.0-0.31                                            ______________________________________                                    

    ______________________________________                                        Eluting System            Rf value                                            ______________________________________                                        chloroform:methanol 85:15 0.51                                                ______________________________________                                    


2. The method of producing antibiotic L 13365 as defined in claim 1which comprises cultivating Actinoplanes sarveparensis CBS No. 305.76 ina culture medium containing an assimilable source of carbon, anassimilable source of nitrogen and inorganic salts under submergedaerobic conditions until a substantial amount of antibiotic L 13365 isproduced by said organism in said culture medium.
 3. The method of claim2 which includes the additional step of isolating antibiotic L 13365 asdefined in claim 2 from said culture medium.